Herbal drug composition for cartilage protection

ABSTRACT

The present invention relates to a herbal drug composition for cartilage protection comprising plant extracts of Clematis Radix, Trichosanthis Radix, and Prunellae Spica and an optimal content of rosmarinic acid to: (i) alleviate pains; (ii) inhibit the acute/chronic inflammation, platelet/whole blood aggregation, immunocyte (B-lymphcyte and T-lymphcyte) proliferation, inflammation-inducing enzyme activities, and enzyme activities associated with degradation of joint tissue; (iii) scavenge activity of toxic active oxygen radicals; and (iv) further provide excellent cartilage protection activity to be effectively used as an anti-inflammatory agent with analgesic effects, blood circulation enhancer, arthritis therapeutic agent and cartilage protective.

FIELD OF THE INVENTION

[0001] The present invention relates to a herbal drug composition forcartilage protection and more particularly, to the drug composition forcartilage protection comprising plant extracts of Clematis Radix,Trichosanthis Radix, and Prunellae Spica and an optimal content ofrosmarinic acid to: (i) alleviate pains; (ii) inhibit the acute/chronicinflammation, platelet/whole blood aggregation, immunocyte (B-lymphocyteand T-lymphocyte) proliferation, inflammation-inducing enzymeactivities, and enzyme activities associated with degradation of jointtissue; (iii) scavenge activity of toxic active oxygen radicals; and(iv) further provide excellent cartilage protection activity to beeffectively used as an anti-inflammatory agent with analgesic effects,blood circulation enhancer, arthritis therapeutic agent and cartilageprotective.

BACKGROUND OF THE INVENTION

[0002] Clematis Radix, Trichosanthis Radix, and Prunellae Spica are wellknown for medicinal plants. Each medicinal plant has long been used forthe treatment of general inflammations, such as various swellings,wounds, bronchitis, mastitis, tonsillitis, and anal fistula and also forthe relief of various symptoms such as cold or numb hands, painfulknees, painful waist and shoulder, feeble in health and pain in theskin, in the form of aqueous plant extract. These symptoms are similarto general arthritis including chronic rheumatism in terms of the modernpathological concept. Clematis Radix, a root of Clematis mandshurica andthe same genera in plant taxonomy, is distributed in the shady forestthroughout Asia. It is collected in autumn, washed cleanly afterremoving cormophyte and root hair, chopped finely and dried in the sunto be used as a medicinal use. Clematis Radix, a non-toxic medicinalplant, has long been used for the treatment of the following symptoms:pains in the extremities; motor disturbance in knee joints; andparalysis in the extremities. In particular, Clematis Radix has beenfrequently used as a miraculous drug in those patients who feeluncomfortable while standing due to the coldness in waist, knees andfeet. It is well known that Clematis Radix has various constituents offlavanone glycosides such as clematin, etc. and saponins such asclemontanoside A, clemontanoside B, clemontanoside C, and clemontanosideS, glucoses, and sterols [Research Archives of Useful Plants Resourcesin Korea, Korea Research Institute of Chemical Technology,pp780-781(1988), 2. An Explanatory Diagram of Korean Medicinal plants,Youngrim Pub., pp489-490(1990)].

[0003] Trichosanthis Radix, known as “multifarious medicine” or“Karokon”, is a non-toxic medicinal herb prepared by collecting roots ofTrichosnathes kirilowii and the same genera in plant taxonomy, which areperennial liana plants, in autumn. The outer shells of cleanly washedroots are removed and the rest of the roots are cut appropriately anddried in the sun for medicinal use. Trichosanthis Radix has been widelyused for excretion of pus, vanishing the boil, detoxification andantipyretic effect and also effective for diseases symptomized bythirst, various swellings, mastitis, and anal fistula. It has beeninvestigated up to now that Trichosanthis Radix contains trichosanthinas proteins, arginine and citruline as amino acids, and palmitic acidand linoleic acid as fatty acids. Recently Trichosanthis Radix is foundto contain bryonolic acid, 4-hydroxybenzoic acid, α-spinastero assterols [Research Archives of Useful Plants Resources in Korea, KoreaResearch Institute of Chemical Technology, pp 354-357(1988), 2. AnExplanatory Diagram of Korean Medicinal Plants, Youngrim Pub.,pp960-963(1990)].

[0004] Prunellae Spica, a flower or upper part of Prunella vulgaris andthe same genera in plant taxonomy, is a non-toxic medicinal herbprepared by collecting the flower, when it is half withered duringsummer, and drying in the sun. Prunellae Spica has been widely used forthe treatment of the following symptoms: chronic swellings, smallpox,acute mastitis and lymphatic tuberculosis. Prunellae Spica is alsoeffective in the destructing lumps (generated in a lower stomach owingto extravasated blood) or others, while treating beriberi and numbnessin the extremities. It has been reported that Prunellae Spica containssaponins such as oleanolic acid glycosides and ursolic acid glycosides,etc, and also contains carotene, vitamin C, vitamin K, tannin, caffeicacid and chlorogenic acid. Rosmarinic acid is also found in PrunellaeSpica [Research Archives of Useful Plant Resources in Korea, KoreaResearch institute of Chemical Technology, pp 480-482(1988); ChemicalResearch for Prunellae Spica, Lee Jak-pyung et al., Bulletin of MedicalCollege in Beijing, 17(4), pp297-299(1985); Pharm. Acta. Helv., 66, No.7, pp185-188(1991)].

[0005] The conventional oriental herbal books (e.g., Dong-Eui-Bo-gam,Hyangyak Gibsung-bang and Kwangjee Beakub) or related literatures referto the medical efficacy of herbs and processes of manufacturing aqueousherbal solutions. However, they only described a single prescription ofeach of these medicinal plants but not a formulation available for themanufacture of aqueous herbal solution from appropriate combinations ofsorted medicinal plants by harvest place and harvest time to control thecontent of active ingredients. Furthermore, these medicinal plants wereprepared by hot water extraction method, and any substances extracted byabove method showed no acquisition of detailed knowledge on biologicallyactive ingredients.

[0006] On the other hand, the inventors of the present invention havedisclosed a process of extraction and purified biologically effectiveingredients from an extract of Clematis Radix, Trichosanthis Radix, andPrunellae Spica in a certain ratio, being useful for alleviatingacute/chronic inflammation; for inhibiting platelet and whole bloodaggregation, enzyme activities associated with degradation of jointtissue, abnormally proliferated immunocytes, and inflammation-inducingenzymes; for scavenging activity of toxic active oxygen radical; andfurther for the treatment of chronic rheumatoid arthritis (U.S. Pat. No.5,910,307). U.S. Pat. No. 5,910,307 is characterized by mixing ClematisRadix, Trichosanthis Radix, and Prunellae Spica in a weight ratio of1:0.5-2:0.5-1.5 and extracting the mixture with water or aqueousalcoholic solution; partitioning with water-saturated n-butanol andconcentrating the alcohol layer under reduced pressure; andconcentrating the result with water under constant boiling andlyophilizing to obtain an extract in powder form.

[0007] In the continuous study, the inventors have realized that it isdifficult to standardize the extracts of Clematis Radix, TrichosanthisRadix, and Prunellae Spica with simple weight ratio since the contentvariation of each ingredient significantly varies with harvest place andharvest time. This further makes it difficult to merchandize suchcombined extracts since it is hard to obtain the reproducibility ofactive ingredients having analgesic and anti-inflammatory effects, bloodcirculation enhancing effect, arthritis therapeutic effect and cartilageprotection. Thereupon, the inventors have made an extensive research tomaximize the pharmacological efficiency of herbal drug composition withthe reproducibility. As a result, we have developed a method to optimizethe efficiency of cartilage protection effect as well as the analgesicand anti-inflammatory effect, blood circulation enhancing effect, andarthritis therapeutic effect by controlling the content of rosmarinicacid in the herbal drug composition, not the extract weight ratio ofClematis Radix, Trichosanthis Radix, and Prunellae Spica. In addition,it was possible to merchandize medicinal herbs with the reproducibility.

[0008] The present invention, an improved invention of U.S. Pat. No.5,910,307, provides significant improvement in merchandizing byoptimizing the herbal extract composition of Clematis Radix,Trichosanthis Radix, and Prunellae Spica with the proper content ofrosmarinic acid, maximizes pharmacological efficiencies over theconventional herbal composition and further provides novel therapeuticeffects.

SUMMARY OF THE INVENTION

[0009] The present invention is to provide a herbal drug compositioncomprising Clematis Radix, Trichosanthis Radix, and Prunellae, whereinthe ingredients extracted and purified are standardized and merchandizedbased on the content of rosmarinic acid for the purpose ofreproducibility and useful for an anti-inflammatory agent with analgesiceffects, blood circulation enhancer, arthritis therapeutic agent andcartilage protective, etc. The herbal drug composition of the presentinvention has similar or superior efficiency for analgesic andanti-inflammatory effect and improving blood circulation to theconventional herbal composition of U.S. Pat. No. 5,910,307. In additionto that, it has excellent inhibitory activities against enzymesassociated with degradation of joint tissue and a protection activitytoward cartilage and thus, it is superiorly effective to generalarthritis including rheumatism.

[0010] Accordingly, the object of the present invention is to provide aherbal drug composition having excellent analgesic and anti-inflammatoryeffect, improving effect of peripheral blood circulation, arthritistherapeutic effect and cartilage protection effect by standardizing theextracts from mixed Clematis Radix, Trichosanthis Radix, and PrunellaeSpica with the proper content of rosmarinic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 represents the inhibitory activity of the herbal drugcomposition on edema induced by carrageenan.

[0012]FIG. 2 represents the anti-coagulant activity of the herbal drugcomposition on platelet aggregation.

[0013]FIG. 3 represents the anti-coagulant activity of the herbal drugcomposition on whole blood aggregation.

[0014]FIG. 4 represents the inhibitory activity of the herbal drugcomposition on chronic inflammation.

[0015]FIG. 5 represents the inhibitory activity of the herbal drugcomposition on B-lymphocyte proliferation.

[0016]FIG. 6 represents the inhibitory activity of the herbal drugcomposition on T-lymphocyte proliferation.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention is characterized by a combined herbal drugcomposition comprising Clematis Radix, Trichosanthis Radix, PrunellaeSpica and more than 0.6 wt. % of rosmarinic acid based on the totalcomposition.

[0018] The present invention is described in detail as set forthhereunder.

[0019] The herbal drug composition of the present invention providessignificant biological effects such as analgesic and anti-inflammations,improvement of blood circulation, immunoregulation and inhibitions onenzymes associated with degradation of joint tissue, and particularly,cartilage protection and thus, being useful as arthritis therapeuticagent and cartilage protective as well as an analgesic andanti-inflammatory agent and blood circulation enhancer.

[0020] The content of active ingredients in herbal drugs variesremarkably with harvest place, harvest time, storage period and storagecondition. Therefore, it is desirable to combine herbal extracts orherbal plants in an appropriate ratio depending on the harvest place asshown in Tables 1 to 3. That is, if harvest place, harvest time, storageperiod and storage condition are different, the content of activeingredients contained in the same weight of herbal plant has differentcontents, thus the limitation with the weight ratio of herbal extractsor herbal plants becomes insignificant.

[0021] Therefore, the present invention is characterized by selectingrosmarinic acid as a reference material to obtain the herbal drugcomposition with optimal pharmacological efficiency. Rosmarinic acid isknown to have excellent activities such as: (i) antioxidant activity byinhibiting lipid peroxidation and/or biosynthesis of prostacyclingenerated in the metabolism of arachidonic acid, and by scavenging theactive oxygen generated from polymorphonuclear leukocytes; (ii)anti-inflammatory activity such as inhibition against the generation ofmetabolites which are carriers for inflammation reaction andimmunoregulation to inhibit allergic inflammation; (iii) bloodcirculation enhancing activity by inhibition of platelet bloodaggregation and fiber degradation [Agent and Action, 17,pp375-376(1985); Pharm. Acta. Helv., 66, No. 7, pp185-188(1991);Biochem. Pharmae., 29, pp533-538(1980); Yoa-Hsueh-Hseuh-Pao, 27,pp96-100(1992); Int. J. of Immunopharmae., 10, No. 6, pp729-737(1988);J. of Natural Products, 50, No. 3, pp392-399(1987); Yoa-Hsueh-Hseuh-Pao,28, No. 4, pp241-245(1993)]. The variation of a rosmarinic acid contentis broad depending on harvest place, harvest time, storage period andstorage condition etc. and further the efficiency varies with thecontent of rosmarinic acid. This is the reason why rosmarinic acid isselected as a reference material in the present invention. Accordingly,the herbal drug composition of the present invention can be prepared bymixing the herbal extracts of Clematis Radix, Trichosanthis Radix,Prunellae Spica or extracting the mixture of Clematis Radix,Trichosanthis Radix, Prunellae Spica not depending on the weight ratioof each herbal plant but depending on the content of rosmarinic acidcontained in the total herbal plants.

[0022] The herbal drug composition can obtain the desired pharmaceuticalefficiencies when the content of rosmarinic acid is higher than 0.6 wt.% of rosmarinic acid, preferably 0.6-5.0 wt. %. When the content ofrosmarinic acid is higher than 0.6 wt. % in the herbal drug compositioncomprising Clematis Radix, Trichosanthis Radix, Prunellae Spica, itprovides optimal effects such as cartilage protection activity which hasnot been taught in the conventional herbal drug compositions as well asalleviation of pains, improvement of blood circulation,immunoregulation, and inhibition of enzymes associated with degradationof joint tissue. If the content of rosmarinic acid is lower than 0.6 wt.%, the herbal drug composition provides alleviation of pains,improvement of blood circulation, immunoregulation, and inhibition ofenzymes associated with degradation of joint tissue but very sluggishcartilage protection activity. The present invention has no upperlimitation for the amount of the rosmarinic acid but when it is higherthan a certain level, the activities are not improved any further, thusit is not desirable technically and economically to increase the contentof rosmarinic acid to higher than a certain level.

[0023] According to the present invention, when the herbal drugcomposition contains 2.0-6.0 wt. % of oleanolic acid and 0.01-0.04 wt. %of 4-hydroxybenzoic acid in addition to higher than 0.6 wt. % ofrosmarinic acid, it provides far more potent efficacy toward cartilageprotection activity as well as alleviation of pains, improvement ofblood circulation, immunoregulation, and inhibition of enzymesassociated with degradation of joint tissue.

[0024] Large quantity of oleanolic acid is present in the extract ofClematis Radix. When the extract is hydrolyzed, sufficient amounts ofoleanolic acid are present as a sapogenin, a sugar-free form ofsaponins. According to an analysis, it is found that oleanolic acid ispresent as saponins bonded to various glycosides. It has been reportedthat oleanolic acid has not only remarkable anti-inflammatory andanalgesic effects but also an excellent effect for chronic rheumatoidarthritis induced by Mycobacterium butyricum [J. of Pharm. Pharmacol.,44, No.5, pp456-458(1992); Chung-Kuo-Li-Hsueh-Pao, 10, No.4,pp381-384(1984); Chem. Pharm, Bull., 28, No.4, pp1183-1188(1980);Biochem. Int., 24, No.5, pp981-990(1991)].

[0025] The extract of Trichosanthis Radix contains various organic acidsincluding 4-hydroxybenzoic acid. 4-hydroxybenzoic acid is known to haveexcellent anti-oxidant activity, anti-inflammatory activity andhormone-like effect in the uterus ablation osteoporotic model andfurther is one of reference materials for the present invention becauseit is maintained in a certain amount regardless of harvest place,harvest time, storage period and storage conditions [Free Radical Biol.& Medcine, 27, No.11/12, pp1427-1436(1999); J. Ethnopharma., 53,11-14(1996); Environ. Res., 75, pp130-134(1997)].

[0026] Rosmarinic acid, oleanolic acid and 4-hydroxybenzoic acidselected as reference materials are active ingredients of herbal drugcomposition obtained from this invention. The efficacy is far morepotent due to remarkable synergic effect when they are combined in acertain ratio based on the contents of active ingredients. Further, inaddition to such active ingredients of the herbal drug composition fromthis invention, other different ingredients cannot be ruled out in thisinvention.

[0027] To contain certain amounts of reference materials, the herbaldrug composition of the present invention is prepared by either mixingeach extract in powder form of Clematis Radix, Trichosanthis Radix, andPrunellae or extracting the mixture of Clematis Radix, TrichosanthisRadix, and Prunellae Spica based on the chemical analysis of originalherbal plants. The method for preparing herbal drug composition makes alittle difference in cartilage protection activity. The method forpreparing herbal drug composition is described in detail as set forthhereunder.

[0028] Each Clematis Radix, Trichosanthis Radix, and Prunellae Spica ora mixture thereof is extracted with 5-10 volumes of water or aqueousalcoholic solution under reflux for 4 to 6 hours and filtered. Thefiltrate is further extracted with 5-10 volumes of water or aqueousalcoholic solution under reflux and filtered. Each extract is combinedand concentrated to dryness. Hence, if a small amount of the solvent isused, the extraction efficiency is low due to the lower solubility ofextract and the difficulty of stirring the extract. In case of using anexcess of the solvent, however, a larger amount of solvent saturatedwith lower alcohol in water has to be evaporated and removed, thereforewhich is uneconomical and difficult in handling. The present inventionperforms a series of extraction steps, i.e., first and secondextraction, because when the extraction is on a large scale, significantlosses are anticipated due to high contents of water in medicinalplants, in spite of effective filtration. So the second extraction isresponsible for preventing the reduced extraction efficiency rather thanthe first extraction only. Further, it is revealed that about 85-95% ofthe total extract amount is obtained through two extractions. It is alsofound that more than three steps of extraction are not to be desirablein economical matter.

[0029] The extracts are filtered and concentrated, and the filtrate ispurified to remove some impurities such as proteins, polysaccharides andfatty acids by extracting with the same amount of lower alcoholsaturated with 3-4 volumes of water. Examples of lower alcohol used inthe present invention include butyl alcohol and propyl alcohol. If theamount of water-saturated lower alcohol is less than that of thefiltrate, a higher concentration of impurities (e.g., polysaccharides,and proteins) having relatively strong polarity causes lowerconcentration of active ingredients in the extracts due to poorseparation of layers.

[0030] After separating the layers, the obtained fractions extractedwith alcohol are concentrated under reduced pressure at 60-70° C. toremove lower alcohol in the sample. Then, the extract is furtherconcentrated 2-3 times under constant boiling with 25-50 volumes ofwater to the total extract amount and followed with another same amountof water for homogeneous suspension. The reason why the residue isconcentrated under constant boiling with water is to control thecontents of remaining lower alcohol so as to use the extracting solutionas pharmaceutical raw materials. The obtained extract is thenlyophilized to give an extract powder.

[0031] The obtained extract powder has significant pharmacologicaleffects, such as analgesic and anti-inflammatory agents, bloodcirculation enhancers, rheumatoid arthritis therapeutic agents, andcartilage protection agents.

[0032] Based on the general manufacturing method, the powdered extractof this invention is formulated in oral or parenteral administrationsuch as tablets, soft gelatin capsules, injection solutions, ointments,and transdermals.

[0033] For human use, the herbal drug composition of this invention canbe administered alone, but will generally be administered in admixturewith a pharmaceutical carrier selected with regard to the intended routeof administration and standard pharmaceutical practice. For example, theherbal drug composition may be administered orally, buccally orsublingually, in the form of tablets containing excipients such asstarch or lactose, or in capsules or ovules either alone or in admixturewith excipients, or in the form of elixirs or suspensions containingflavoring or coloring agents. Such liquid preparations may be preparedwith pharmaceutically acceptable additives such as suspending agent(e.g., methylcellulose, a semi-synthetic glyceride such as witepsol ormixtures of glycerides such as a mixture of apricot kernel oil and PEG-6esters or mixtures of PEG-8 and caprylic/capric glycerides). A compoundmay also be injected parenterally, for example intravenously,intramuscularly, subcutaneously or intracoronarily. For parenteraladministration, the compound is best used in the form of a sterileaqueous solution that may contain other substances, for example salts,or monosaccharides such as mannitol or glucose, to make the solutionisotonic with blood.

[0034] For administration to man, the effective dose of the herbal drugcomposition may vary with the age, weight, physical condition andresponse of the particular patient and administration may be made once aday or a few times a day according to the physician. Preferred dosagesfor an average adult patient (70 Kg) are as follows: in the range offrom 50 to 2400 mg daily for oral administration such as individualtablets or capsules; in the range of from 50 to 400 mg daily forparenteral administration; in the range of from 300 to 2400 mg daily forointments; and in the range of from 150 to 1200 mg daily for transdermaladministration. The above dosages are exemplary of the average case butthere can be individual instances in which higher or lower dosage rangesmay be merited, and such are within the scope of this invention.Further, if the daily dosages of the herbal drug composition are lessthan the above ranges, the desired effects are not obtained. On theother hands, if they are higher than the above ranges, it is also notpreferable due to toxic side effects.

[0035] In particular, while the herbal drug composition of the presentinvention was administered to human, the toxic side effect is far lessthan other chemically synthesized drugs. As a matter of fact, severaltoxicological tests reveal that the combined extract of the presentinvention is not toxic to the human.

[0036] The present invention is explained in more detail with referenceto the following examples that should not be taken to limit the scope ofthe invention.

REFERENCE EXAMPLE 1

[0037] 250 g of well air dried Clematis Radix where debris were removedby washing with water was extracted from 2 l of 30% (v/v)ethanol-containing water under reflux for 6 hrs while stirring. Afterfiltration, the residue was extracted again from 1.5 l of 30% (v/v)ethanol-containing water under reflux for 6 hrs while stirring. Thefiltrates were combined and concentrated to have 1 l of the totalvolume. The filtrate was extracted with a same volume of water-saturatedn-butanol three times. The n-butanol layers were combined andconcentrated under reduced pressure at 60-70° C. to dryness. 0.3 l ofwater was added to the residue and extracted under constant boiling andrepeated the procedure twice. The extract was well suspended in the sameamount of distilled water and lyophilized to give powdered extract.

[0038] Other Clematis Radix harvested in different places were preparedby the above procedure and analyzed by high performance liquidchromatography. The result was summarized in Table 1 with the content(%) of oleanolic acid. TABLE 1 Harvest Content of Yield place (China)oleanolic acid (%) (%, w/w) Heilongjiang A 6.98 3.25 Heilongjiang B13.59 3.95 Heilongjiang C 2.68 2.76 Jilin A 9.29 2.58 Jilin B 0.12 4.05Jilin C 8.96 2.94 Liaoning A 8.53 3.08 Liaoning B 6.75 2.83 Liaoning C3.97 1.79 Sichuan A 0.15 3.64 Sichuan B 6.98 2.36

REFERENCE EXAMPLE 2

[0039]250 g of well air dried Trichosanthis Radix where debris wereremoved by washing with water was extracted from 2.5 l of 30% (v/v)ethanol-containing water under reflux for 4 hrs while stirring. Afterfiltration, the residue was extracted again from 1.5 l of water underreflux for 3 hrs while stirring. The filtrates were combined andconcentrated to have 1 l of the total volume. The filtrate was extractedwith a same volume of water-saturated n-butanol three times. Then-butanol layers were combined and concentrated under reduced pressureat 60-70° C. to dryness. 0.2 l of water was added to the residue andextracted under constant boiling and repeated the procedure twice. Theextract was well suspended in the same amount of distilled water andlyophilized to give powdered extract.

[0040] Other Trichosanthis Radix harvested in different places wereprepared by the above procedure and analyzed by high performance liquidchromatography. The result was summarized in Table 2 with the content(%) of rosmarinic acid. TABLE 2 Harvest Content of Yield place (China)rosmarinic acid (%) (%, w/w) Henan A 3.13 2.17 Henan B 12.05 2.28 HenanC 8.34 2.06 Hubei A 1.89 2.40 Hubei B 7.22 3.20 Hubei C 5.75 1.52 HunanA 10.96 2.74 Hunan B 4.89 1.92 Hunan C 13.00 3.19 Sichuan A 6.14 1.32Sichuan B 12.45 1.40 Sichuan C 1.59 2.82

REFERENCE EXAMPLE 3

[0041]250 g of well air dried Prunellae Spica having a length of 2.0-4.0cm where debris were removed by washing with water was extracted from 2l of water under reflux for 5 hrs while stirring. After filtration; theresidue was extracted again from 2 l of water under reflux for 3 hrswhile stirring. The filtrates were combined and concentrated to have 1 lof the total volume. The filtrate was extracted with a same volume ofwater-saturated n-butanol three times. The n-butanol layers werecombined and concentrated under reduced pressure at 60-70° C. todryness. 0.1 l of water was added to the residue and extracted underconstant boiling and repeated the procedure twice. The extract was wellsuspended in the same amount of distilled water and lyophilized to givepowdered extract.

[0042] Other Trichosanthis Radix harvested in different places wereprepared by the above procedure and analyzed by high performance liquidchromatography. The result was summarized in Table 3 with the content(%) of 4-hydroxybenzoic acid. TABLE 3 Harvest Content of place4-hydroxybenzoic Yield (China) acid (%) (%, w/w) Hebei A 0.018 0.95Hebei B 0.024 0.76 Hebei C 0.037 0.82 Henan A 0.047 0.91 Henan B 0.0581.05 Henan C 0.019 0.064 Anhui A 0.041 0.71 Anhui B 0.058 1.23 Anhui C0.098 0.86 Zhejiang A 0.045 0.79 Zhejiang B 0.073 1.08 Zhejiang C 0.0621.19

EXAMPLE 1 Preparation of a Mixture of the Herbal Drug Extracts

[0043] Clematis Radix, Trichosanthis Radix, and Prunellae Spica having1.5% (w/w) of rosmarinic acid, 3.5% (w/w) of oleanolic acid, and 0.02%(w/w) of 4-hydroxybenzoic acid were mixed and n-butanol fractionationwas performed three times, respectively. The extract obtained each stepwas combined and well suspended in the same amount of distilled waterand lyophilized to give powdered extract.

EXAMPLE 2 Preparation of a Mixture of the Herbal Drug Extracts

[0044] Clematis Radix, Trichosanthis Radix, and Prunellae Spica having0.7% (w/w) of rosmarinic acid, 5.0% (w/w) of oleanolic acid, and 0.01%(w/w) of 4-hydroxybenzoic acid were mixed and n-butanol fractionationwas performed three times, respectively. The extract obtained each stepwas combined and well suspended in the same amount of distilled waterand lyophilized to give powdered extract.

EXAMPLE 3 Preparation of a Mixture of the Herbal Drug Extracts

[0045] Clematis Radix, Trichosanthis Radix, and Prunellae Spica having4.0% (w/w) of rosmarinic acid, 2.0% (w/w) of oleanolic acid, and 0.04%(w/w) of 4-hydroxybenzoic acid were mixed and n-butanol fractionationwas performed three times, respectively. The extract obtained each stepwas combined and well suspended in the same amount of distilled waterand lyophilized to give powdered extract.

EXAMPLE 4 Preparation of a Herbal Extract of Mixed Plants

[0046] 200 g of well dried Clematis Radix harvested in Heilongjiang Awhere debris were removed by washing with water, 450 g of TrichosanthisRadix harvested in Henan A with a length of 2.0-4.0 cm, and 350 g ofPrunellae Spica harvested in Henan B were mixed and the mixture wasextracted with 10 l of water under reflux for 6 hrs. After filtration,the residue was extracted again from 7 l of water under reflux for 3 hrswhile stirring. The filtrates were combined and concentrated to give 5 lof the total volume. The filtrate was extracted with the same volume ofwater-saturated n-butanol three times. The n-butanol layers werecombined and concentrated under reduced pressure at 60-70° C. todryness. One liter of water was added to the residue and extracted underconstant boiling and repeated the procedure twice. The extract was wellsuspended in the same amount of distilled water and lyophilized to givepowdered extract.

[0047] The powdered extract was determined by HPLC analysis having 2.1%(w/w) of rosmarinic acid, 2.1% (w/w) of oleanolic acid, and 2.5% (w/w)of 4-hydroxybenzoic acid.

COMPARATIVE EXAMPLE 1

[0048] Well dried Clematis Radix harvested in Heilongjiang B wheredebris were removed by washing with water, 500 g of Trichosanthis Radixharvested in Anhui C with a length of 2.0-4.0 cm, and 250 g of PrunellaeSpica harvested in Hubei A were mixed and the mixture was extracted with15 l of water under reflux for 6 hrs. After filtration, the residue wasextracted again with 7 l of water under reflux for 3 hrs while stirring.The filtrates were combined and concentrated to give 5 l of the totalvolume. The filtrate was fractionated with the same volume ofwater-saturated n-butanol three times. The n-butanol layers werecombined and concentrated under reduced pressure at 60-70° C. todryness. One liter of water was added to the residue and extracted underconstant boiling and the procedure was repeated twice. The extract waswell suspended in the same amount of distilled water and lyophilized togive powdered extract.

[0049] The powdered extract was determined by HPLC analysis having 0.4%(w/w) of rosmarinic acid, 6.8% (w/w) of oleanolic acid, and 0.05% (w/w)of 4-hydroxybenzoic acid.

COMPARATIVE EXAMPLE 2 Preparation of a Mixture of Herbal Drug Extract

[0050] Each extract of Clematis Radix, Trichosanthis Radix, andPrunellae Spica prepared in Reference Examples 1, 2, and 3 was mixed anddissolved in aqueous alcoholic solution to have the contents of 0.45%(w/w) of rosmarinic acid, 6.11% (w/w) of oleanolic acid, and 0.02% (w/w)of 4-hydroxybenzoic acid. The mixture was concentrated under reducedpressure. The extract was well suspended in the same amount of distilledwater and lyophilized to give powdered extract.

EXPERIMENTAL EXAMPLE 1 Test for Analgesic Effects

[0051] To investigate the analgesic effects of various extracts preparedby said Examples 1-4 and Comparative Examples 1-2, writhing test wasconducted as presented in the following experimental method and theresult was expressed as Table 4.

[0052] Experimental Method:

[0053] The herbal extracts, prepared by said Examples 1-4 andComparative Examples 1-2, were orally administered to ICR (Institute ofCancer Research) mice at doses of 200 mg or 400 mg per Kg of bodyweight.

[0054] One hour after administration, 0.6% (v/v) acetic acid wasintraperitoneally injected to the animals at a volume of 0.1 ml per 10 gof body weight and from 5 minutes after administration, writhingfrequency of each mice was observed for 10 minutes as a pain threshold.TABLE 4 Dose of herbal Avg. Rate of drug extract writhing inhibitionCategory (mg/Kg) frequency (%) Control — 19 — Example 1 200 12 36.8 4009 52.6 Example 2 200 11 42.1 400 8 57.9 Example 3 200 11 42.1 400 9 52.6Example 4 200 10 47.4 400 7 63.2 Comparative 200 13 31.6 Example 1 40010 47.4 Comparative 200 14 26.3 Example 2 400 11 42.1

[0055] According to the Table 4, it is revealed that the extractprepared by the present invention has superior analgesic effects fromreduced writhing frequencies.

EXPERIMENTAL EXAMPLE 2 Test for Inhibitory Activity on AcuteInflammation

[0056] The inhibitory activity of the herbal extracts, prepared by saidExamples 1-4 and Comparative Examples 1-2, on acute inflammation wasinvestigated in rats. In comparison with the control, theanti-inflammatory effect on hind paw in edema was expressed as percentand the result is presented in the attached FIG. 1.

[0057] Experimental Method:

[0058] The herbal extracts, prepared by said Examples 1-4 andComparative Examples 1-2, was orally administered to white SD(Spraque-Dawley) rats. At one hour after administration, 0.1 ml of 1%carrageenan was intradermally injected to the left hind paw of rats andedema at that site was measured at 1 hour interval for 5 hours.

[0059] As noted in the attached in FIG. 1, it is revealed that theherbal extracts prepared by said Examples 1-4 of the present inventionsignificantly inhibited the carrageenan-induced inflammation.

EXPERIMENTAL EXAMPLE 3 Test for the Anti-Aggregating Activity

[0060] The anti-coagulant activity of the herbal extracts, prepared bysaid Examples 1-4 and Comparative Examples 1-2, was investigated inrabbits plasma and the aggregation was induced by collagen and theresult is presented in the attached FIG. 2.

[0061] Experimental Method:

[0062] PRP (platelet rich plasma) was prepared from the blood sample ofrabbits and the number of platelet in blood was adjusted at 2×10⁸/ml.The herbal extracts, prepared by said Examples 1-4 and ComparativeExamples 1-2, were added to the PRP and adjusted on a cuvette ofaggregometer at 37° C. for 2 minutes. With the addition of collagenplatelet aggregation was measured with a dual aggregometer.

[0063] As noted in the attached FIG. 2, there was no increase inplatelet aggregation by the herbal.

EXPERIMENTAL EXAMPLE 4 Test for the Anti-Coagulant Activity on WholeBlood Coagulation

[0064] The anti-coagulant activity of the herbal extracts, prepared bysaid Examples 1-4 and Comparative Examples 1-2, was investigated usingwhole blood of rabbit and the result is presented in the attached FIG.3.

[0065] Experimental Method:

[0066] The same amount of saline solution was added to whole blood ofrabbit and mixed well prior to use in this experiment. The herbalextracts, prepared by said Examples 1-4 and Comparative Examples 1-2,were added to reconstituted whole blood and prewormed for 2 minutes.And, the blood coagulation was induced by the addition of collagen. Thewhole blood coagulation was measured by an aggregometer. As noted in theattached FIG. 3, there was no increase in whole blood coagulation whenthe herbal extracts prepared by said Examples 1-4 of the presentinvention was added.

EXPERIMENTAL EXAMPLE 5 Test for the Inhibitory Activity onHyaluronidase, an Enzyme Associated with Degradation of Joint Tissue

[0067] The inhibitory activity of the herbal extracts, prepared by saidExamples 1-4 and Comparative Examples 1-2, on hyaluronidase, an enzymeassociated with degradation of joint tissue, was investigated and theresult is presented in the following Table 5.

[0068] Experimental Method:

[0069] Hyaluronidase was prepared in the presence of acetate buffersolution at 37° C. for 20 minutes and activated. Then the herbalextracts, prepared by said Examples 1-4 and Comparative Examples 1-2,and potassium hyaluonate as a substrate were added to the buffersolution and cultured for about 40 minutes. After terminating thereaction with sodium hydroxide, potassium borate was added to thecultures and heated at 100° C. The absorbance was measured by thedevelopment of DMBA (dimethylbenzanthracene) and the rate of inhibitionwas calculated in comparison with control. TABLE 5 Test Rate ofconcentration inhibition Category (mg/ml) (%) Example 1 1 82.3 Example 21 83.7 Example 3 1 89.6 Example 4 1 87.5 Comparative Example 1 1 80.5Comparative Example 2 1 79.1

[0070] As shown in Table 5, the combined herbal extracts prepared by thepresent invention significantly inhibited the activation of the enzymeassociated with degradation of joint tissue.

EXPERIMENTAL EXAMPLE 6 Test for the Inhibitory Activity on ChronicInflammation

[0071] The anti-inflammatory activity of the herbal extracts, preparedby said Examples 1-4 and Comparative Examples 1-2, on chronic rheumatoidarthritis was investigated in Mycobacterium butyricum injected ratmodels. The result is presented in the attached FIG. 4.

[0072] Experimental Method:

[0073] To induce chronic edema, Mycobacterium butyricum suspended inmineral oil and treated with heat was injected to the right hind paw ofwhite rats at each dose of 0.05 ml. Then, the herbal extracts, preparedby said Examples 1-4 and Comparative Examples 1-2, were orallyadministered to the rats once daily for 16 days, and the paw edema wasmeasured with plethysmometer.

[0074] As shown in the attached FIG. 4, the combined herbal extracts bythe present invention significantly inhibited the edema.

EXPERIMENTAL EXAMPLE 7 Test for the Inhibitory Activity on LeukotrieneB₄

[0075] The inhibitory activity of the herbal extracts, prepared by saidExamples 1-4 and Comparative Examples 1-2, on 5-lipoxygenase wasevaluated by the inhibition rate of leukotriene B₄ (LTB₄) biosynthesisinduced by arachidonic acid and calcium ionophore (A23187) and theresult is presented in the Table 6.

[0076] Experimental Method:

[0077] The extracts, prepared by said Examples 1-4 and ComparativeExamples 1-2, were added to rat basophilic leukemia-1 (RBL-1) cellsadjusted at 37° C. and reacted for 5 minutes. Then the reacting mixturewas treated with 20 μg/ml A23187 and arachidonic acid for 15 minutes soas to induce the generation of LTB₄. The generated LTB₄ was extractedwith ethyl acetate and was subjected to HPLC. TABLE 6 Test Rate ofconcentration inhibition Category (mg/ml) (%) Example 1 0.5 87.3 Example2 0.5 82.5 Example 3 0.5 79.1 Example 4 0.5 85.7 Comparative Example 10.5 71.5 Comparative Example 2 0.5 70.6

[0078] As shown in the Table 6, the combined herbal extracts prepared bythe present invention significantly inhibited 5-lipoxygenase.

EXPERIMENTAL EXAMPLE 8 Test for the Inhibitory Activity onCyclooxygenase-I

[0079] The inhibitory activity of the herbal extracts, prepared by saidExamples 1-4 and Comparative Examples 1-2 on cyclooxygenase-I wasevaluated by arachidonic acid and the result is presented in the Table7.

[0080] Experimental Method:

[0081] The extracts, prepared by said Examples 1-4 and ComparativeExamples 1-2, were added to cyclooxygenase-I adjusted at 37° C., and 100μM arachidonic acid was added and reacted for 2 minutes, trichloroaceticacid (TCA) was added to the reacting mixture for terminating thereaction and absorbance was measured at 530 nm. TABLE 7 Test Rate ofconcentration inhibition Category (mg/ml) (%) Example 1 0.5 61.8 Example2 0.5 62.5 Example 3 0.5 59.8 Example 4 0.5 67.3 Comparative Example 10.5 51.5 Comparative Example 2 0.5 49.1

[0082] As shown in the Table 7, the combined herbal extracts prepared bythe present invention significantly inhibited cyclooxygenase-I.

EXPERIMENTAL EXAMPLE 9 Test for the Inhibitory Activity onCyclooxygenase-II

[0083] The inhibitory activity of the extracts, prepared by saidExamples 1-2 and 5 Comparative Examples 1-3, on cyclooxygenase-II wasevaluated and the result is presented in the Table 8.

[0084] Experimental Method:

[0085] The extracts prepared by said Examples 1-2 and ComparativeExamples 1-3, were added to cyclooxygenase-II and placed at a test tubeadjusted at 27° C. After reaction with 500 mM arachidonic acid for 90seconds, trichloroacetic acid (TCA) was added to the reaction mixturefor terminating the reaction and absorbance was measured at 532 nm.TABLE 8 Test Rate of concentration inhibition Category (mg/ml) (%)Example 1 0.5 72.5 Example 2 0.5 69.8 Example 3 0.5 65.7 Example 4 0.577.5 Comparative Example 1 0.5 61.5 Comparative Example 2 0.5 58.8

[0086] As shown in the Table 8, it is noted that the combined plantextracts prepared by this invention significantly inhibitedcyclooxygenase-II.

EXPERIMENTAL EXAMPLE 10 Test for the Inhibitory Activity on theProliferation of B-Lymphocyte

[0087] The inhibitory activity of the herbal extracts, prepared by saidExamples 1-4 and Comparative Examples 1-2 on the proliferation ofB-lymphocyte induced by lipopolysaccharide (LPS) was evaluated and theresult is presented in the attached FIG. 5.

[0088] Experimental Method:

[0089] Cultures were set up with 10⁶ B-lymphocyte/ml of medium at 37° C.The extracts, prepared by said Examples 1-4 and Comparative Examples1-2, were added to the cultures, and then the cultures were treated with10 μg/ml of LPS for 24 hours. With the addition of 2 μCi Thymidine-³Hexpressed by tritium as radioactivity for 48 hours, cultures werequantified on liquid scintillation counter (LSC).

[0090] As shown in the attached FIG. 5, it is noted that the combinedherbal extracts prepared by the present invention significantlyinhibited the proliferation of B-lymphocyte.

EXPERIMENTAL EXAMPLE 11 Test for the Inhibitory Activity on theProliferation of T-Lymphocyte

[0091] The inhibitory activity of the herbal extracts, prepared by saidExamples 1-4 and Comparative Examples 1-2, on the proliferation ofT-lymphocyte induced by concanavalin-A (Con-A) were investigated and theresult is presented in the attached FIG. 6.

[0092] Experimental Method:

[0093] Cultures were set up with 10⁶ T-lymphocyte/ml of medium at 37° C.The extracts, prepared by said Examples 1-4 and Comparative Examples1-2, were added to the cultures, which were treated with 3 μg/ml ofconcanavalin-A for 24 hours. With the addition of 2 μCi Thymidine-³Hexpressed by tritium as radioactivity for 48 hours, cultures werequantified on LSC.

[0094] As shown in the attached FIG. 6, it is noted that the combinedherbal extracts prepared by the present invention significantlyinhibited the proliferation of T-lymphocyte.

EXPERIMENTAL EXAMPLE 12 Test for the Scavenging Activity on Eliminationof Superoxide Radicals

[0095] The scavenging activity of the herbal extracts, prepared by saidExamples 1-4 and Comparative Examples 1-2, was assessed on theelimination of superoxide radicals generated from xanthine-xanthineoxidase and the result is presented in the Table 9.

[0096] Experimental Method:

[0097] Cytochrome-c (Cyt-c) and herbal extracts, prepared by Examples1-4 and Comparative Examples 1-2, were added to xanthine oxidaseadjusted at 37° C. so as to induce the generation of oxygen radicals byxanthine. The changes in color along with oxidation of cytochrome-c(Cyt-c) was measured by spectrophotometer at 540 nm and scavenging rateof oxygen radicals was also measured as slope. TABLE 9 Test Rate ofconcentration inhibition Category (mg/ml) (%) Example 1 0.5 82.7 Example2 0.5 89.5 Example 3 0.5 83.6 Example 4 0.5 92.1 Comparative Example 10.5 81.5 Comparative Example 2 0.5 78.4

[0098] As shown in the Table 9, it is noted that the combined herbalextracts prepared by the present invention significantly scavengedactive oxygen.

EXPERIMENTAL EXAMPLE 13 Test for the Inhibitory Activity on ProteoglycanDegradation

[0099] The inhibitory activity of the herbal extracts, prepared by saidExamples 1-4 and Comparative Examples 1-2, on the Proteoglycandegradation which is an important element in cartilage tissue wasinvestigated and the result is presented in the Table 10.

[0100] Experimental Method:

[0101] Cartilage from rabbits was dissected from each joint and 50 mg ofcartilage was placed in a culture medium adjusted at 37° C. The herbalextract, prepared by said Examples 1-4 and Comparative Examples 1-2, and5 μg of interleukin-1α (IL-1α) per 50 mg of cartilage were added to theculture medium and cultured for 72 hours. The production ofglucosaminoglycan (GAG), which is produced by proteoglycan degradation,was measured at 525 nm by coloring with 1,9-dimethylmethylene blue dye.TABLE 10 Test Rate of inhibition conc. on the proliferation of Category(mg/ml) glucosaminoglycan (%) Example 1 1.0 115 0.3 83 0.1 68 Example 21.0 103 0.3 91 0.1 79 Example 3 1.0 99 0.3 81 0.1 73 Example 4 1.0 1210.3 94 0.1 75 Comparative 1.0 84 Example 1 0.3 52 0.1 38 Comparative 1.081 Example 2 0.3 46 0.1 37

[0102] As shown in the Table 10, it is noted that the combined herbalextracts prepared by the present invention significantly inhibited theproteoglycan degradation activity.

EXPERIMENTAL EXAMPLE 14 Test for the Inhibitory Activity on Type IICollagen Degradation

[0103] The inhibitory activity of the herbal extracts, prepared by saidExamples 1-4 and Comparative Examples 1-2, on the Type II collagendegradation which is an important element in articular cartilage tissuewas investigated and the result is presented in the Table 11.

[0104] Experimental Method:

[0105] Cartilage from rabbits was dissected from each joint and 50 mg ofcartilage was placed in a culture medium adjusted at 37° C. The herbalextract, prepared by said Examples 1-4 and Comparative Examples 1-2, and5 μg of interleukin-lα (IL-1α) per 50 mg of cartilage were added to theculture medium and cultured for 21 days. The production of hydroxyproline, which is produced by type II collagen degradation, was measuredat 560 nm by coloring with chloramines-T and dimethylaminobenzaldehyde.TABLE 11 Rate of inhibition on the proliferation Category Test conc.(mg/ml) of hydroxy-Proline (%) Example 1 1.0 91 0.3 63 0.1 38 Example 21.0 89 0.3 59 0.1 39 Example 3 1.0 97 0.3 76 0.1 53 Example 4 1.0 93 0.365 0.1 46 Comparative 1.0 67 Example 1 0.3 36 0.1 19 Comparative 1.0 65Example 2 0.3 36 0.1 20

[0106] As shown in the Table 11, it is noted that the combined herbalextracts prepared by the present invention showed more significantinhibitory activity on the Type II collagen degradation than that ofComparative Example 1.

EXPERIMENTAL EXAMPLE 15 Test for the Therapeutic Efficacy AgainstOsteoarthritis

[0107] The activity on oateoarthritis of the herbal extracts, preparedby said Examples 1-4 and Comparative Examples 1-2, on cartilageprotection was investigated by monitoring rabbit models of whichcartilage was injured by injecting collagenase to cavum articular ofrabbit to have similar symptoms to human osteoarthritis. The result ispresented in the Table 12.

[0108] Experimental Method:

[0109] 1 mg of collagenase per 1 Kg of body weight was injected at thefirst and fourth days to the cavum articular of rabbit having 2.0-2.5 Kgof body weight to injure joint tissue and to have collagenase-inducedarthritis. The extracts, prepared by said example 1-4 and comparativeexample 1-2, were given orally at a dose of 200 mg/Kg for 4 weeks. After4 weeks of administration, the cartilage part was dissected andwithdrawn from the rabbit and then dyed with sapranin-O. The dyedcartilage part was divided into cartilaginous tissue and synovial tissueand the degree of arthritis severity was recorded with the integer scaleof 0-4 to quantify the levels: 0=normal; 1=slight; 2=moderate; 3=severe;and 4=maximum. TABLE 12 Cartilaginous Category tissue¹⁾ Synovialtissue²⁾ Total³⁾ Control 11.8 10.8 21.6 Example 1 6.0 6.5 12.5 Example 26.2 7.2 13.4 Example 3 6.3 7.1 13.4 Example 4 5.0 6.0 11.0 Comparative8.6 8.2 16.8 Example 1 Comparative 8.7 8.0 16.7 Example 2

[0110] As shown in the Table 12, it is noted that the combined herbalextracts prepared by the present invention showed more significantalleviation activity on collagenase-induced arthritis than that ofComparative Example 1.

MANUFACTURING EXAMPLE 1

[0111] The following chemical composition was employed for themanufacture of oral tablets using the powdered extracts of the presentinvention.

[0112] Chemical Composition: the herbal drug composition 200 mg; hardanhydrous silicate 10 mg; Magnesium stearate 2 mg; microcrystallinecellulose 50 mg; Sodium starch glycolate 25 mg; corn starch 113 mg; andanhydrous ethanol in an appropriate amount.

MANUFACTURING EXAMPLE 2

[0113] The following chemical composition was employed for themanufacture of ointments using the powdered extracts of the presentinvention.

[0114] Chemical Composition: the herbal drug composition 5 g; cetylpalmitate 20 g; cetyl alcohol 40 g; stearyl alcohol 20 g; isopropylmyristate 80 g; sorbitan monostearate 20 g; polysorbate 60 g; propylp-hydroxybenzoate 1 g; methyl p-hydroxybenzoate 1 g; and phosphoric acidand distilled water in an appropriate amount.

MANUFACTURING EXAMPLE 3

[0115] The following chemical composition was employed for themanufacture of injection solutions using the powdered extracts of thepresent invention.

[0116] Chemical Composition: the herbal drug composition 100 mg;mannitol 180 mg; Na₂HPO₄ 25 mg; and water for injection 2,974 mg.

MANUFACTURING EXAMPLE 4

[0117] The following chemical composition was employed for themanufacture of transdermal using the powdered extracts of the presentinvention.

[0118] Chemical Composition 1: the herbal drug composition 0.4 g;poly(acrylic acid, sodium salt) 1.3 g; glycerin 3.6 g; aluminumhydroxide 0.04 g; methyl paraben 0.2 g; and water 14 g.

[0119] Chemical Composition 2: the herbal drug composition 0.8 g;propylene glycol 1.6 g; fluid paraffin 0.8 g;

[0120] Several dosage forms (e.g., tablets, ointments, transdermal andinjection solutions) prepared by said manufacture 1-4 related tocombined herbal preparations using Clematis Radix, Trichosabthis Radixand Prunellae Spica according to this invention. Said preparationscontain concentrations of oleanolic acid and rosmarinic acid asreference materials, thus being effectively used for anti-inflammatoryagent with analgesic effects, chronic rheumatoid arthritis drug andagent for improving peripheral blood circulation.

What is claimed is:
 1. A herbal drug composition for the cartilageprotection comprising Clematis Radix, Trichosanthis Radix, PrunellaeSpica, wherein more than 0.6 wt. % of rosmarinic acid is present in thetotal composition.
 2. The herbal drug composition for the cartilageprotection according to claim 1, wherein said rosmarinic acid is presentin the range of from 0.6 to 5.0 wt. % relative to the total composition.3. The herbal drug composition for the cartilage protection according toclaim 1, wherein said herbal drug composition comprises oleanolic acidand 4-hydroxybenzoic acid in addition to said rosmarinic acid.
 4. Theherbal drug composition for the cartilage protection according to claim1, wherein said oleanolic acid is present in the range of from 2.0 to6.0 wt. % to the total composition.
 5. The herbal drug composition forthe cartilage protection according to claim 3, wherein said4-hydroxybenzoic acid is present in the range of from 0.01 to 0.04 wt. %to the total composition.
 6. A cartilage protective comprising theherbal drug composition selected from the possible combinationsconsisting of said claims 1-5 as an active ingredient for cartilageprotection and arthritis therapeutic treatment.
 7. The cartilageprotective according to claim 6, wherein said cartilage protective isformulated in the form of tablets or soft gelatin capsules for oraladministration, injection solutions, ointments, or transdermals.
 8. Thecartilage protective according to claim 7, wherein the dosage of saidtablet or soft gelatin capsule for oral administration is in the rangeof from 50 to 2400 mg daily.